This grant is aimed at understanding how transcription factor codes pattern the retina and control guidance receptor expression to establish the crossed and uncrossed visual projections. Our previous work defined such a program for the ipsilateral projection through the mouse optic chiasm: The transcription factor Zic2 and the guidance receptor EphB1 are expressed in ventrotemporal (VT) retinal ganglion cells (RGCs), which give rise to the ipsilateral projection. EphrinB2 is expressed on radial glia at the optic chiasm midline, and the repulsive EphB1-ephrinB2 interaction produces the ipsilateral projection. Zic2 and EphB1 expression correlate with the degree of binocularity across species and in genetic models with a reduced ipsilateral projection, such as the albino. In the last funding period, we determined that EphB1 expression is downregulated in Zic2 mutants and upregulated after ectopic expression of Zic2;thus, Zic2 controls EphB1 expression. Further, Zic2 is necessary and sufficient to drive an ipsilateral projection in vivo and in vitro. We also found that Foxd1 is expressed in the VT quadrant and is required for Zic2 and EphB1 expression, placing Foxd1 upstream of this transcriptional program for the ipsilateral projection. In other studies, we found that the Ig-CAM NrCAM and the Semaphorin receptor PlexinA1 are expressed by RGCs in non-VT and late-forming VT retina, both regions giving rise to the contralateral projection. NrCAM appears to modulate an inhibitory response by RGCs to semaphorins, as found for midline crossing in other systems. In Aim 1, we will continue a focus on Zic2 and identify additional genes regulated by Zic2. The transcription factor Islet2 has an identical expression pattern to NrCAM and PlexinA1, but its role is restricted to the late-born crossed projection from VT, similar to NrCAM. Therefore, we will focus on the late VT retina and determine whether Islet2 controls NrCAM and/or PlexinA1 expression to encode the contralateral retinal projection from this region. We will also determine if Islet2 interacts functionally with Zic2. In Aim 2, we will search for additional genes that may specify the retinal sectors giving rise to the crossed and uncrossed projection, and investigate whether Foxg1 (the nasal counterpart to Foxd1) is upstream of the contralateral program. In Aim 3, we will apply information gained from Aims 1 and 2 to understand how the the albino retina is (mis)specified to produce a diminished ipsilateral projection. Aim 4 will examine whether the genes important for RGC and retinal specification implement midline guidance and/or targeting of eye- specific zones in the dorsal lateral geniculate nucleus (dLGN). These studies use innovative methods for gene delivery (in utero and ex vivo electroporation), in vitro assays, and circuit tracing to define the molecular control of the formation of binocular visual projections. PUBLIC HEALTH RELEVANCE: This research aims to understand how retinal ganglion cells grow out from each eye, meet at the X-shaped optic chiasm, then diverge toward targets on both sides of the brain. Proper binocular vision is dependent on a normal distribution of retinal axons crossing at the optic chiasm, and if altered, reduced visual acuity and depth perception ensue. This work investigates the genes that pattern the retina into sectors giving rise to crossed and uncrossed projections and that drive expression of guidance receptors to enable retinal axons to take the appropriate route.